Optimized Preparation of Whole Murine Tumor-Bearing Lung Tissue for Flow Cytometry and Single-Cell RNA-Sequencing
May 22nd, 2026
This protocol presents a detailed method for isolating high-quality single-cell suspensions from both healthy and tumor-bearing murine lung tissue. This protocol can be applied to scRNA-seq, flow cytometry analysis, and primary cell isolation.
Our research investigates how circadian clocks control biological process and how their disruption promotes tumorgenesis. To overcome limited subpopulation analysis, we develop this protocol to sequence all cell types within tumor-bearing lungs together. To begin, gather the required reagents, prepare DMEM/F-12 medium by adding 2%volume per volume heat inactivated FBS to the medium.
Prepare the cell wash buffer by adding heat inactivated FBS to calcium and magnesium-free PBS. Pre-chill calcium and magnesium-free PBS to use as the perfusion buffer. Next, prepare the given enzyme stock solutions.
Then combine them as shown to prepare the digestion medium. Prepare the resuspension buffer by adding one microliter of 0.5 molar EDTA and one milliliter of heat inactivated FBS to 50 milliliters of calcium and magnesium-free PBS. After placing the euthanized mouse on its back on a dissection pad, pin the feet, spray the mouse liberally with 70%ethanol.
Using disinfected surgical instruments, make a two to three centimeter wide midline incision through the skin and muscles of the lower abdomen. Carefully open the thorax without cutting the lungs or the heart. Attach a safety multifly 21 gauge needle to a 50 milliliter syringe.
Aspirate 30 milliliters of perfusion buffer into the syringe. Insert the needle into the left ventricle using the sharp straight scissors. Make a small incision in the right atrium.
Perfuse the whole body with 15 milliliters of perfusion buffer until the liver is whitened. Remove the lungs from the thorax using scissors. Cut away the excess tissue attached to the lungs.
Place the lungs in 10 milliliters of pre-cool calcium and magnesium-free PBS to wash away excess blood and maintain the sample on ice between each step throughout the experiment. Transfer the lungs into a 1.5 milliliter tube containing 500 microliters of ice cold DMEM/F-12 medium. Using an extra narrow straight scissors, cut the lung tissue into small pieces of one to two cubic millimeters for two to three minutes.
Transfer the minced lung tissue to a six centimeter dish containing five milliliters of digestion Medium. Place the six centimeter dish on a thermostatic shaker set to 50 revolutions per minute for 25 minutes at 37 degrees Celsius. Using a one milliliter pipette, mix the suspension every eight minutes.
Wet the 100 micrometer mesh strainer with one milliliter of DMEM/F-12 medium and place it in a 10 centimeter dish. Transfer the digested suspension into the strainer. Using the rubber end of a sterile syringe plunger, push the tissue through the strainer.
Place a 70 micrometer mesh strainer over a 50 milliliter centrifuge tube and pre-wet it with one milliliter of DMEM/F-12 medium. Filter the cell suspension through the strainer. Rinse the strainer with five milliliters of DMEM/F-12 medium.
Centrifuge the filtered cell suspension at 400 x g for 10 minutes at four degrees Celsius. After removing the supernatant, resuspend the cell pellet in one milliliter of red blood cell lysing buffer for two minutes at room temperature. Add 14 milliliters of cell wash buffer to the suspension.
After centrifuging as previously demonstrated, discard the supernatant. Once the pellet appears clear, resuspend the cells in 20 milliliters of resuspesion buffer. Mix 10 microliters of the single cell suspension with an equal volume of trypan blue.
Add 10 microliters of the mixture to the cell counting chamber slide. Check live cell viability, cell number, and single cell status using a cell counter. Cell viability was below 80%before optimization and increased to above 85%after optimization.
Cell ranger quality assessment identified 23, 488 estimated cells in sample one and 21, 436 in sample two. The barcode rank plot indicated minimal ambient ribonucleic acid contamination. Principle component analysis and uniform manifold approximation and projection revealed well-defined clusters with all major lung cell types represented.
Dot plots of representative marker genes showed that canonical biomarkers were highly expressed in their corresponding cell types. Tdtomato-positive cells were enriched in epithelial-related clusters. Tdtomato and epithelial cell adhesion molecule-positive cells were successfully isolated using flow cytometry.
This protocol allows researchers to study the heterogeneity in various murine lung models, including tumor-bearing whole lungs. The protocol also enables downstream analysis, such as cell-cell communication mapping to further characterize the tumor microenvironment. Future studies could investigate how circadian resume disruptions promotes tumorgenesis through cell-cell communication and microenvironment remodeling.
Overview
This article presents a detailed protocol for isolating high-quality single-cell suspensions from both healthy and tumor-bearing mouse lung tissue, specifically in a KrasG12D; tdTomato reporter mouse model. The method is optimized to achieve over 85% cell viability, enabling robust single-cell RNA sequencing (scRNA-seq) analysis of lung tissue heterogeneity, including during tumorigenesis.
Key Study Components
Area of Science
- Cancer biology
- Single-cell genomics
- Lung tissue analysis
Background
- Non-small cell lung cancer (NSCLC) constitutes about 85% of lung cancers, with KRAS as the most frequently mutated gene.
- Tumor heterogeneity and plasticity in NSCLC are not fully understood.
- scRNA-seq is essential for dissecting cellular diversity in complex tissues like the lung.
- Obtaining viable single-cell suspensions from fibrotic, tumor-bearing lung tissue is technically challenging.
Purpose of Study
- To develop and optimize a protocol for isolating viable single cells from healthy and tumor-bearing mouse lungs.
- To enable comprehensive scRNA-seq analysis of all cell types within tumor-bearing lungs.
- To facilitate downstream studies of tumor microenvironment and cell-cell communication.
Methods Used
- Preparation of specialized media and buffers for tissue processing.
- Perfusion of mouse lungs to remove blood and minimize contamination.
- Controlled enzymatic digestion and gentle mechanical dissociation of lung tissue.
- Sequential filtration through 100 μm and 70 μm mesh strainers.
- Red blood cell lysis and multiple wash steps to purify single cells.
- Viability assessment using trypan blue exclusion and automated cell counting.
- Fluorescence-activated cell sorting (FACS) to isolate tdTomato+ tumor cells.
Main Results
- Cell viability improved from below 80% to above 85% after protocol optimization.
- scRNA-seq quality assessment identified over 21,000–23,000 estimated cells per sample with minimal ambient RNA contamination.
- Principal component analysis and UMAP revealed well-defined clusters representing all major lung cell types.
- tdTomato+ tumor cells were efficiently enriched and detected in epithelial-related clusters.
Conclusions
- The protocol enables high-quality single-cell isolation from both healthy and tumor-bearing mouse lungs.
- It supports comprehensive scRNA-seq analysis, facilitating studies of lung tumor heterogeneity and microenvironment.
- The method is adaptable for investigating cell-cell communication and the effects of circadian disruption on tumorigenesis.
